Patterning Genes in Retinal Development ? Project Summary The long-range goal of this grant is to determine the cellular and molecular events that lead to the generation of specific cell types in the vertebrate retina. In this renewal we focus upon mechanisms regulating differential expression of the cone visual pigment genes on tandemly-replicated gene arrays. In humans a tandemly-replicated array on the X chromosome consists of one long wavelength-sensitive (LWS) gene followed by 1-9 medium wavelength-sensitive (MWS) genes, which have diverged spectrally from LWS. This recent replication has provided most humans with trichromatic color vision, because LWS, MWS, and SWS1 opsins are uniquely expressed in separate cone populations. Heritable defects in the LWS/MWS array result in various forms of color blindness, X-linked retinal degenerations, and Bornholm Eye Disease, a cone dysfunction associated with high myopia. Insights into regulation of differential expression of LWS vs. MWS opsins could allow therapeutic manipulation of gene expression to treat these disorders. In addition, future regenerative approaches to the treatment of other retinal disorders that involve the loss of cones (age-related macular degeneration; Stargardt?s disease) would ideally include similar in vitro or in vivo manipulations to generate cone phenotypes in ratios that support high-acuity color vision. The widely-accepted model for human LWS vs. MWS opsin regulation states that a stochastic event favors an association of an upstream regulatory region with the LWS or most proximal MWS. However, topographic patterns of the LWS:MWS ratio suggest that a nonrandom, trans regulatory mechanism may be involved. Pursuit of regulatory mechanisms has been challenging because within mammals, only primate genomes contain tandem opsin arrays. In contrast, the genomes of teleost fish, including zebrafish, contain numerous tandem arrays of opsins, which are the consequences of independent gene replication events and neofunctionalization. In our published and preliminary data we demonstrate that in zebrafish, the developmental signaling molecules retinoic acid (RA), and thyroid hormone (T3) can each control differential expression of the tandem duplicates, LWS1 vs. LWS2, an array orthologous to the human LWS/MWS array. Furthermore, compelling preliminary data suggest that RA can promote expression of LWS opsin in human iPSC-derived 3D retinal organoids, changing the ratio of LWS:MWS. Together these findings lay the groundwork for a tremendous breakthrough in understanding determination of LWS vs. MWS cone subtype. In this renewal we pursue mechanisms through which RA and T3 control differential expression of tandemly replicated opsins, and apply this knowledge to retinal regeneration and human retinal organoid development, with three Specific Aims: 1. Determine the relative roles of RA and T3 signaling and their receptors as endogenous regulators of differential expression of tandemly replicated opsin genes. 2. Determine mechanisms through which RA and T3 signaling regulate tandemly replicated opsin genes. 3. Determine roles of RA and T3 signaling for controlling cone fates during retinal regeneration and in human iPSC-derived 3D retinal organoids. These studies will uncover novel mechanisms for the differential expression of tandemly replicated opsin genes, and will generate information necessary to manipulate cone phenotypic fates in concert with the application of regenerative or cell replacement therapies for human retinal degenerations.